def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, sample_node, reference_node = antecedents
fastq_files = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
ref = alignlib.create_reference_genome(reference_node.identifier)
assert os.path.exists(ref.fasta_file_full)
filelib.safe_mkdir(out_path)
metadata = {}
metadata["tool"] = "bowtie2 %s" % alignlib.get_bowtie2_version()
# Make a list of the jobs to run.
jobs = []
for x in fastq_files:
sample, pair1, pair2 = x
sam_filename = os.path.join(out_path, "%s.sam" % sample)
log_filename = os.path.join(out_path, "%s.log" % sample)
x = sample, pair1, pair2, sam_filename, log_filename
jobs.append(x)
sq = mlib.sq
commands = []
for x in jobs:
sample, pair1, pair2, sam_filename, log_filename = x
nc = max(1, num_cores / len(jobs))
x = alignlib.make_bowtie2_command(ref.fasta_file_full,
pair1,
fastq_file2=pair2,
sam_file=sam_filename,
num_threads=nc)
x = "%s >& %s" % (x, sq(log_filename))
commands.append(x)
metadata["commands"] = commands
metadata["num_cores"] = num_cores
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
x = [x[-2] for x in jobs]
filelib.assert_exists_nz_many(x)
return metadata
def set_out_attributes(self, antecedents, out_attributes):
#import os
#from genomicode import config
#from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils
group_node, fastq_node, reference_node = antecedents
fastq_files = module_utils.find_merged_fastq_files(
group_node.identifier, fastq_node.identifier)
assert fastq_files, "No fastq files."
ref = alignlib.create_reference_genome(reference_node.identifier)
# Possibilities:
# 1. All single.
# 2. All paired.
# 3. Mixed.
attrs = out_attributes.copy()
all_pair2 = [x[-1] for x in fastq_files]
uniq_pair2 = {}.fromkeys(all_pair2).keys()
if uniq_pair2 == [None]:
# All single.
attrs["orientation"] = "single"
return attrs
if None in all_pair2:
# Mixed.
raise AssertionError, "Mixed single and paired-end."
# All paired.
# Optimization: check just the first group of FASTQ files and
# assume they all have the same orientation.
sample, pair1_filename, pair2_filename = fastq_files[0]
x = get_paired_orientation(
ref.fasta_file_full, pair1_filename, pair2_filename)
orient, reads_ns, reads_fr, reads_rf, reads_ff = x
#orientation = "paired"
#if x:
# orientation = "paired_%s" % x
#attrs["orientation"] = orientation
attrs["orientation"] = "paired_%s" % orient
key = (group_node.identifier, fastq_node.identifier,
reference_node.identifier)
self.cache[key] = reads_ns, reads_fr, reads_rf, reads_ff
return attrs
def run(self, network, antecedents, out_attributes, user_options,
num_cores, outfile):
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, group_node, reference_node = antecedents
fastq_files = mlib.find_merged_fastq_files(group_node.identifier,
fastq_node.identifier)
assert fastq_files, "No fastq files."
ref = alignlib.create_reference_genome(reference_node.identifier)
metadata = {}
orientation = None
reads_ns = reads_fr = reads_rf = reads_ff = None
# Possibilities:
# 1. All single.
# 2. All paired.
# 3. Mixed. (not handled)
all_pair2 = [x[-1] for x in fastq_files]
uniq_pair2 = {}.fromkeys(all_pair2).keys()
if uniq_pair2 == [None]:
# All single.
orientation = "single"
elif None in all_pair2:
# Mixed.
raise AssertionError, "Mixed single and paired-end."
else:
# All paired.
# Optimization: check just the first group of FASTQ files and
# assume they all have the same orientation.
sample, pair1_filename, pair2_filename = fastq_files[0]
x = get_paired_orientation(ref.fasta_file_full, pair1_filename,
pair2_filename)
orient, reads_ns, reads_fr, reads_rf, reads_ff = x
orientation = "paired_%s" % orient
assert orientation
x = mlib.Orientation(orientation, reads_ns, reads_fr, reads_rf,
reads_ff)
mlib.write_orientation(x, outfile)
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import filelib
from genomicode import alignlib
from genomicode import parallel
from genomicode import hashlib
from Betsy import module_utils as mlib
fastq_node, sample_node, strand_node, reference_node = antecedents
fastq_files = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
assert fastq_files, "I could not find any FASTQ files."
ref = alignlib.create_reference_genome(reference_node.identifier)
stranded = mlib.read_stranded(strand_node.identifier)
filelib.safe_mkdir(out_path)
metadata = {}
metadata["tool"] = "RSEM %s" % alignlib.get_rsem_version()
# Figure out whether to align to genome or transcriptome.
x = out_attributes["align_to"]
assert x in ["genome", "transcriptome"]
align_to_genome = (x == "genome")
# RSEM makes files:
# <sample_name>.genome.bam
# <sample_name>.transcript.bam
# <sample_name>.genes.results
# <sample_name>.isoforms.results
# <sample_name>.stat
#
# Does not work right if there is a space in the sample name.
# Therefore, give a hashed sample name, and then re-name
# later.
# Make a list of the jobs to run.
jobs = []
for x in fastq_files:
sample, pair1, pair2 = x
sample_h = hashlib.hash_var(sample)
x1, x2, x3 = mlib.splitpath(pair1)
x = "%s%s" % (hashlib.hash_var(x2), x3)
pair1_h = os.path.join(out_path, x)
if pair2:
x1, x2, x3 = mlib.splitpath(pair2)
x = "%s%s" % (hashlib.hash_var(x2), x3)
pair2_h = os.path.join(out_path, x)
results_filename = os.path.join(out_path,
"%s.genes.results" % sample)
log_filename = os.path.join(out_path, "%s.log" % sample)
x = filelib.GenericObject(sample=sample,
sample_h=sample_h,
pair1=pair1,
pair2=pair2,
pair1_h=pair1_h,
pair2_h=pair2_h,
results_filename=results_filename,
log_filename=log_filename)
jobs.append(x)
# Make sure hashed samples are unique.
seen = {}
for j in jobs:
assert j.sample_h not in seen, \
"Dup (%d): %s" % (len(jobs), j.sample_h)
assert j.pair1_h not in seen
assert j.pair2_h not in seen
seen[j.sample_h] = 1
seen[j.pair1_h] = 1
seen[j.pair2_h] = 1
# Symlink the fastq files.
for j in jobs:
os.symlink(j.pair1, j.pair1_h)
if j.pair2:
os.symlink(j.pair2, j.pair2_h)
s2fprob = {
"unstranded": None,
"firststrand": 0.0,
"secondstrand": 1.0,
}
assert stranded.stranded in s2fprob, "Unknown stranded: %s" % \
stranded.stranded
forward_prob = s2fprob[stranded.stranded]
# How much memory for bowtie. May need to increase this if
# there are lots of memory warnings in the log files:
# Warning: Exhausted best-first chunk memory for read
# ST-J00106:110:H5NY5BBXX:6:1101:18203:44675 1:N:0:1/1
# (patid 2076693); skipping read
# Default is 64.
# Seems like too high a value can cause problems.
#chunkmbs = 4*1024 # Generates warnings.
chunkmbs = 512
# Get lots of warnings with bowtie:
# Warning: Detected a read pair whose two mates have different names
# Use STAR aligner instead.
use_STAR = True
sq = parallel.quote
commands = []
for j in jobs:
# Debug: If the results file exists, don't run it again.
if filelib.exists_nz(j.results_filename) and \
filelib.exists(j.log_filename):
continue
# If using the STAR aligner, then most memory efficient
# way is to let STAR take care of the multiprocessing.
nc = max(1, num_cores / len(jobs))
if use_STAR:
nc = num_cores
keywds = {}
if use_STAR:
keywds["align_with_star"] = True
else:
keywds["align_with_bowtie2"] = True
x = alignlib.make_rsem_command(ref.fasta_file_full,
j.sample_h,
j.pair1_h,
fastq_file2=j.pair2_h,
forward_prob=forward_prob,
output_genome_bam=align_to_genome,
bowtie_chunkmbs=chunkmbs,
num_threads=nc,
**keywds)
x = "%s >& %s" % (x, sq(j.log_filename))
commands.append(x)
metadata["commands"] = commands
metadata["num cores"] = num_cores
# Need to run in out_path. Otherwise, files will be everywhere.
nc = num_cores
if use_STAR:
nc = 1
parallel.pshell(commands, max_procs=nc, path=out_path)
# Rename the hashed sample names back to the original unhashed
# ones.
files = os.listdir(out_path)
rename_files = [] # list of (src, dst)
for j in jobs:
if j.sample == j.sample_h:
continue
for f in files:
if not f.startswith(j.sample_h):
continue
src = os.path.join(out_path, f)
x = j.sample + f[len(j.sample_h):]
dst = os.path.join(out_path, x)
rename_files.append((src, dst))
for src, dst in rename_files:
filelib.assert_exists(src)
os.rename(src, dst)
# Delete the symlinked fastq files.
for j in jobs:
filelib.safe_unlink(j.pair1_h)
filelib.safe_unlink(j.pair2_h)
# Make sure the analysis completed successfully.
x1 = [x.results_filename for x in jobs]
x2 = [x.log_filename for x in jobs]
filelib.assert_exists_nz_many(x1 + x2)
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, sample_node, ref_node = antecedents
fastq_files = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
ref = alignlib.create_reference_genome(ref_node.identifier)
filelib.safe_mkdir(out_path)
# Do a quick check to make sure the reference is correct.
# Otherwise, error may be hard to disgnose.
alignlib.assert_is_STAR_reference(ref.path)
metadata = {}
metadata["tool"] = "STAR %s" % alignlib.get_STAR_version()
# Figure out the strandedness.
is_stranded = False
# STAR --runThreadN 40 --genomeDir test05 \
# --readFilesIn test.fastq/test03_R1_001.fastq \
# test.fastq/test03_R2_001.fastq --outFileNamePrefix test06.
# If unstranded, add --outSAMstrandField intronMotif
# Make a list of the jobs to run.
jobs = [] # list of filelib.GenericObject objects
for x in fastq_files:
sample, pair1, pair2 = x
out_prefix = "%s." % sample
bam_filename = os.path.join(out_path,
"%sAligned.out.bam" % out_prefix)
log_filename = os.path.join(out_path, "%s.log" % sample)
x = filelib.GenericObject(
sample=sample,
pair1=pair1,
pair2=pair2,
out_prefix=out_prefix,
bam_filename=bam_filename,
log_filename=log_filename,
)
jobs.append(x)
# Run pass 1.
commands = []
for j in jobs:
x = os.path.join(out_path, j.out_prefix)
cmd = alignlib.make_STAR_command(ref.path, x, num_cores,
is_stranded, j.pair1, j.pair2,
j.log_filename)
# For debugging. If this file already exists, skip it.
if not filelib.exists_nz(j.bam_filename):
parallel.sshell(cmd, path=out_path)
filelib.assert_exists_nz(j.bam_filename)
commands.append(cmd)
metadata["commands"] = commands
metadata["num_cores"] = num_cores
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, sample_node, strand_node, ref_node = antecedents
fastq_files = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
ref = alignlib.create_reference_genome(ref_node.identifier)
stranded = mlib.read_stranded(strand_node.identifier)
filelib.safe_mkdir(out_path)
# Do a quick check to make sure the reference is correct.
# Otherwise, error may be hard to disgnose.
alignlib.assert_is_STAR_reference(ref.path)
metadata = {}
metadata["tool"] = "STAR %s" % alignlib.get_STAR_version()
x = mlib.get_user_option(user_options,
"two_pass",
allowed_values=["no", "yes"])
two_pass = (x == "yes")
# Figure out the strandedness.
is_stranded = stranded.stranded != "unstranded"
# STAR --runThreadN 40 --genomeDir test05 \
# --readFilesIn test.fastq/test03_R1_001.fastq \
# test.fastq/test03_R2_001.fastq --outFileNamePrefix test06.
# If unstranded, add --outSAMstrandField intronMotif
# Make a list of the jobs to run.
jobs = [] # list of filelib.GenericObject objects
for x in fastq_files:
sample, pair1, pair2 = x
pass1_out_prefix = "p1.%s." % sample
pass2_out_prefix = "%s." % sample
pass1_bam_filename = os.path.join(
out_path, "%sAligned.out.bam" % pass1_out_prefix)
pass2_bam_filename = os.path.join(
out_path, "%sAligned.out.bam" % pass2_out_prefix)
sjdb_filename = os.path.join(out_path, "p1.%s.SJ.out.tab" % sample)
log1_filename = os.path.join(out_path, "p1.%s.log" % sample)
log2_filename = os.path.join(out_path, "%s.log" % sample)
x = filelib.GenericObject(
sample=sample,
pair1=pair1,
pair2=pair2,
pass1_out_prefix=pass1_out_prefix,
pass2_out_prefix=pass2_out_prefix,
pass1_bam_filename=pass1_bam_filename,
pass2_bam_filename=pass2_bam_filename,
sjdb_filename=sjdb_filename,
log1_filename=log1_filename,
log2_filename=log2_filename,
)
jobs.append(x)
# Run pass 1.
commands = []
for j in jobs:
x = os.path.join(out_path, j.pass1_out_prefix)
cmd = alignlib.make_STAR_command(ref.path, x, num_cores,
is_stranded, j.pair1, j.pair2,
j.log1_filename)
# For debugging. If this file already exists, skip it.
if not filelib.exists_nz(j.pass1_bam_filename):
parallel.sshell(cmd, path=out_path)
filelib.assert_exists_nz(j.pass1_bam_filename)
commands.append(cmd)
if two_pass:
# Make a new index with the splice junction information.
sj_index = os.path.join(out_path, "genome.2pass")
x = [x.sjdb_filename for x in jobs]
filelib.assert_exists_nz_many(x)
x = alignlib.make_STAR_index_command(ref.fasta_file_full,
sj_index,
sjdb_files=x,
num_cores=num_cores)
x = "%s >& genome.2pass.log" % x
commands.append(x)
# For debugging. If this file already exists, skip it.
if not filelib.exists_nz("genome.2pass.log"):
parallel.sshell(x, path=out_path)
alignlib.assert_is_STAR_reference(sj_index)
# Run pass 2.
for j in jobs:
# For debugging. If this file already exists, skip it.
if os.path.exists(j.pass2_bam_filename):
continue
if two_pass:
x = os.path.join(out_path, j.pass2_out_prefix)
cmd = alignlib.make_STAR_command(sj_index, x, num_cores,
is_stranded, j.pair1, j.pair2,
j.log2_filename)
parallel.sshell(cmd, path=out_path)
commands.append(cmd)
else:
# link pass1_bam_filename to pass2_bam_filename
os.symlink(j.pass1_bam_filename, j.pass2_bam_filename)
continue
filelib.assert_exists_nz(j.pass2_bam_filename)
metadata["commands"] = commands
metadata["num_cores"] = num_cores
# STAR takes 28 Gb per process. Make sure we don't use up
# more memory than is available on the machine.
# Defaults:
# --limitGenomeGenerateRAM 31000000000
# --outFilterMismatchNmax 10 Num mismatches.
#nc = mlib.calc_max_procs_from_ram(50, buffer=100, upper_max=num_cores)
#metadata["num_cores"] = nc
#parallel.pshell(commands, max_procs=nc, path=out_path)
# Make sure the analysis completed successfully.
#x = [x[-2] for x in jobs] # sam_filename
#filelib.assert_exists_nz_many(x)
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_filename):
import os
from genomicode import filelib
from genomicode import parallel
from Betsy import module_utils as mlib
fastq_node, sample_node, align_node = antecedents
fastq_data = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
assert fastq_data, "I could not find any FASTQ files."
align_filenames = filelib.list_files_in_path(align_node.identifier,
endswith=".matches.txt")
assert align_filenames, "No .matches.txt files."
align_filenames.sort()
metadata = {}
assert len(fastq_data) == len(align_filenames), \
"Mismatch: num samples %d %d" % (
len(fastq_data), len(align_filenames))
num_mismatches = mlib.get_user_option(user_options,
"num_mismatches",
type=int)
assert num_mismatches >= 0 and num_mismatches < 25
metadata["num_mismatches"] = num_mismatches
sample2fastqdata = {}
for x in fastq_data:
sample, f1, f2 = x
sample2fastqdata[sample] = x
# list of (sample, align_filename, summary_filename,
# fastq_filename1, fastq_filename2)
jobs = []
for in_filename in align_filenames:
p, f = os.path.split(in_filename)
# <sample>.matches.txt
ext = ".matches.txt"
assert f.endswith(ext)
sample = f[:-len(ext)]
assert sample in sample2fastqdata, "Missing FASTQ: %s" % sample
summary_filename = "%s.summary.txt" % sample
x, fastq_filename1, fastq_filename2 = sample2fastqdata[sample]
x = sample, in_filename, summary_filename, \
fastq_filename1, fastq_filename2
jobs.append(x)
jobs2 = [] # list of (function, args, keywds)
for x in jobs:
sample, align_filename, summary_filename, \
fastq_file1, fastq_file2 = x
args = align_filename, fastq_file1, fastq_file2, num_mismatches
keywds = {
"temp_path": ".",
"outfile": summary_filename,
}
x = summarize_matches_file, args, keywds
jobs2.append(x)
# Since this can take a lot of memory (depending on the number
# of reads, can easily take 8 Gb), do just 1 process at a
# time. Also, I/O intensive. Don't do too many at a time.
#MAX_PROCS = 1
MAX_PROCS = 4
nc = mlib.calc_max_procs_from_ram(30, upper_max=MAX_PROCS)
#nc = min(MAX_PROCS, num_cores)
results = parallel.pyfun(jobs2, num_procs=nc, DELAY=0.1)
metadata["num_cores"] = nc
assert len(results) == len(jobs2)
# Put together the results in a table.
handle = open(out_filename, 'w')
header = "sample", "match", "total", "RPM", "match", "mismatch"
print >> handle, "\t".join(header)
for x in zip(jobs, results):
x, d = x
sample, in_filename, summary_filename, \
fastq_filename1, fastq_filename2 = x
match = d["perfect_alignments"]
total = d["total_alignments"]
rpm = int(float(match) / total * 1E6)
perc_match = d["perc_perfect"]
perc_mismatch = 1 - d["perc_perfect"]
x = sample, match, total, rpm, perc_match, perc_mismatch
assert len(x) == len(header)
print >> handle, "\t".join(map(str, x))
handle.close()
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, outfile):
from genomicode import parselib
from genomicode import parallel
from Betsy import module_utils as mlib
MAX_CORES = 4 # I/O intensive.
fastq_node, sample_node, bam_node = antecedents
bam_filenames = mlib.find_bam_files(bam_node.identifier)
sample2fastq = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier,
as_dict=True)
metadata = {}
jobs = [] # list of (sample, bam_file, fastq_file)
for filename in bam_filenames:
path, sample, ext = mlib.splitpath(filename)
assert sample in sample2fastq, "Missing fastq: %s" % sample
fastq1, fastq2 = sample2fastq[sample]
x = sample, filename, fastq1
jobs.append(x)
funcalls = []
for x in jobs:
sample, bam_filename, fastq_filename = x
# Count the number of reads.
x1 = count_reads, (fastq_filename, ), {}
# Count the number of alignments.
x2 = count_alignments, (bam_filename, ), {}
funcalls.append(x1)
funcalls.append(x2)
assert len(funcalls) == len(jobs) * 2
nc = min(num_cores, MAX_CORES)
results = parallel.pyfun(funcalls, num_procs=nc)
metadata["num_cores"] = nc
# list of (sample, aligns, aligned_reads, total_reads, perc_aligned).
results2 = []
for i, x in enumerate(jobs):
sample, bam_filename, fastq_filename = x
x1 = results[i * 2]
x2 = results[i * 2 + 1]
total_reads = x1
aligned_reads, alignments = x2
perc_aligned = float(aligned_reads) / total_reads
x = sample, alignments, aligned_reads, total_reads, perc_aligned
results2.append(x)
results = results2
# sort by sample name
results.sort()
# Make table where the rows are the samples and the columns
# are the statistics.
table = []
header = ("Sample", "Alignments", "Aligned Reads", "Total Reads",
"Perc Aligned")
table.append(header)
for x in results:
sample, alignments, aligned_reads, total_reads, perc_aligned = x
x1 = parselib.pretty_int(alignments)
x2 = parselib.pretty_int(aligned_reads)
x3 = parselib.pretty_int(total_reads)
x4 = "%.2f%%" % (perc_aligned * 100)
x = sample, x1, x2, x3, x4
assert len(x) == len(header)
table.append(x)
# Write out the table as text file.
TXT_FILE = "summary.txt"
handle = open(TXT_FILE, 'w')
for x in table:
print >> handle, "\t".join(x)
handle.close()
txt2xls = mlib.findbin("txt2xls", quote=True)
parallel.sshell("%s -b %s > %s" % (txt2xls, TXT_FILE, outfile))
return metadata
def run(
self, network, antecedents, out_attributes, user_options, num_cores,
out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils
fastq_node, group_node, reference_node = antecedents
fastq_path = fastq_node.identifier
assert os.path.exists(fastq_path)
assert os.path.isdir(fastq_path)
ref = alignlib.create_reference_genome(reference_node.identifier)
filelib.safe_mkdir(out_path)
#reference_fa = module_utils.find_bwa_reference(index_path)
metadata = {}
metadata["tool"] = "bwa %s" % alignlib.get_bwa_version()
# Find the merged fastq files.
x = module_utils.find_merged_fastq_files(
group_node.identifier, fastq_path)
grouped_fastq_files = x
# Make sure no duplicate samples.
x1 = [x[0] for x in grouped_fastq_files]
x2 = {}.fromkeys(x1).keys()
assert len(x1) == len(x2), "dup sample"
# Make a list of all the jobs to do.
jobs = [] # list of (sample, pair1, pair2, bam_filename)
for x in grouped_fastq_files:
sample, pair1, pair2 = x
bam_filename = os.path.join(out_path, "%s.bam" % sample)
log_filename = os.path.join(out_path, "%s.log" % sample)
x = sample, pair1, pair2, bam_filename, log_filename
jobs.append(x)
# Uses ~6 Gb per process.
# Calculate the number of cores per job.
nc = max(1, num_cores/len(jobs))
metadata["num cores"] = nc
# Make the bwa commands.
commands = []
for x in jobs:
sample, pair1, pair2, bam_filename, log_filename = x
x = alignlib.make_bwa_mem_command(
ref.fasta_file_full, log_filename, pair1,
fastq_file2=pair2, bam_filename=bam_filename, num_threads=nc)
commands.append(x)
metadata["commands"] = commands
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
x1 = [x[-2] for x in jobs]
x2 = [x[-1] for x in jobs]
filelib.assert_exists_nz_many(x1 + x2)
return metadata
def run(
self, network, antecedents, out_attributes, user_options, num_cores,
out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, sample_node, strand_node, reference_node, gene_node = \
antecedents
fastq_files = mlib.find_merged_fastq_files(
sample_node.identifier, fastq_node.identifier)
ref = alignlib.create_reference_genome(reference_node.identifier)
assert os.path.exists(ref.fasta_file_full)
gtf_file = gene_node.identifier
filelib.assert_exists_nz(gtf_file)
stranded = mlib.read_stranded(strand_node.identifier)
filelib.safe_mkdir(out_path)
metadata = {}
metadata["tool"] = "TopHat %s" % alignlib.get_tophat_version()
# Get the GTF file, if any.
#gtf_file = module_utils.get_user_option(
# user_options, "tophat_gtf_file", check_file=True)
transcriptome_fa = mlib.get_user_option(
user_options, "tophat_transcriptome_fa", check_file=True)
assert gtf_file or transcriptome_fa, (
"Either tophat_gtf_file or tophat_transcriptome_fa (preferred) "
"must be provided.")
# Make a list of the jobs to run.
jobs = []
for x in fastq_files:
sample, pair1, pair2 = x
tophat_path = os.path.join(out_path, "%s.tophat" % sample)
log_filename = os.path.join(out_path, "%s.log" % sample)
x = sample, pair1, pair2, tophat_path, log_filename
jobs.append(x)
# Generate tophat commands for each of the files.
s2ltype = {
"unstranded" : "fr-unstranded",
"firststrand" : "fr-firststrand",
"secondstrand" : "fr-secondstrand",
}
assert stranded.stranded in s2ltype, "Unknown stranded: %s" % \
stranded.stranded
library_type = s2ltype[stranded.stranded]
# Takes ~3 Gb per process.
sq = parallel.quote
commands = []
for x in jobs:
sample, pair1, pair2, tophat_path, log_filename = x
nc = max(1, num_cores/len(jobs))
x = alignlib.make_tophat_command(
ref.fasta_file_full, tophat_path, pair1, fastq_file2=pair2,
gtf_file=gtf_file, transcriptome_fa=transcriptome_fa,
library_type=library_type, num_threads=nc)
x = "%s >& %s" % (x, sq(log_filename))
commands.append(x)
metadata["commands"] = commands
metadata["num_cores"] = num_cores
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
x = [x[3] for x in jobs] # out_path
x = [os.path.join(x, "accepted_hits.bam") for x in x]
bam_filenames = x
filelib.assert_exists_nz_many(bam_filenames)
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, sample_node, orient_node, reference_node = antecedents
fastq_files = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
ref = alignlib.create_reference_genome(reference_node.identifier)
assert os.path.exists(ref.fasta_file_full)
orient = mlib.read_orientation(orient_node.identifier)
filelib.safe_mkdir(out_path)
metadata = {}
metadata["tool"] = "bowtie1 %s" % alignlib.get_bowtie1_version()
# With low alignment percentages, might want to play around with:
# - insert size
# - maximum mismatch
# Make a list of the jobs to run.
jobs = []
for x in fastq_files:
sample, pair1, pair2 = x
sam_filename = os.path.join(out_path, "%s.sam" % sample)
log_filename = os.path.join(out_path, "%s.log" % sample)
x = sample, pair1, pair2, sam_filename, log_filename
jobs.append(x)
# Generate bowtie1 commands for each of the files.
attr2orient = {
"single": None,
"paired_fr": "fr",
"paired_rf": "rf",
"paired_ff": "ff",
}
orientation = attr2orient[orient.orientation]
#x = sample_node.data.attributes["orientation"]
#orientation = attr2orient[x]
sq = parallel.quote
commands = []
for x in jobs:
sample, pair1, pair2, sam_filename, log_filename = x
nc = max(1, num_cores / len(jobs))
x = alignlib.make_bowtie1_command(ref.fasta_file_full,
sam_filename,
pair1,
fastq_file2=pair2,
orientation=orientation,
num_threads=nc)
x = "%s >& %s" % (x, sq(log_filename))
commands.append(x)
metadata["commands"] = commands
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
for x in jobs:
sample, pair1, pair2, sam_filename, log_filename = x
# Make sure sam file created.
assert filelib.exists_nz(sam_filename), \
"Missing: %s" % sam_filename
# Make sure there are some alignments.
x = open(log_filename).read()
assert x.find("No alignments") < 0, "No alignments"
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from genomicode import hashlib
from Betsy import module_utils as mlib
fastq_node, sample_node, orient_node, reference_node = antecedents
fastq_files = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
ref = alignlib.create_reference_genome(reference_node.identifier)
assert os.path.exists(ref.fasta_file_full)
orient = mlib.read_orientation(orient_node.identifier)
filelib.safe_mkdir(out_path)
metadata = {}
metadata["tool"] = "bowtie2 %s" % alignlib.get_bowtie2_version()
# Bowtie2 doesn't handle files with spaces in them. Make
# temporary files without spaces.
# Make a list of the jobs to run.
jobs = []
for i, x in enumerate(fastq_files):
sample, pair1, pair2 = x
bam_filename = os.path.join(out_path, "%s.bam" % sample)
log_filename = os.path.join(out_path, "%s.log" % sample)
sample_h = hashlib.hash_var(sample)
temp_pair1 = "%d_%s_1.fa" % (i, sample_h)
temp_pair2 = None
if pair2:
temp_pair2 = "%d_%s_2.fa" % (i, sample_h)
j = filelib.GenericObject(sample=sample,
pair1=pair1,
pair2=pair2,
temp_pair1=temp_pair1,
temp_pair2=temp_pair2,
bam_filename=bam_filename,
log_filename=log_filename)
jobs.append(j)
for j in jobs:
os.symlink(j.pair1, j.temp_pair1)
if pair2:
os.symlink(j.pair2, j.temp_pair2)
# Generate bowtie2 commands for each of the files.
attr2orient = {
"single": None,
"paired_fr": "fr",
"paired_rf": "rf",
"paired_ff": "ff",
}
orientation = attr2orient[orient.orientation]
#x = sample_node.data.attributes["orientation"]
#orientation = attr2orient[x]
# Takes ~4 Gb per job.
samtools = mlib.findbin("samtools")
sq = parallel.quote
commands = []
for j in jobs:
#sample, pair1, pair2, bam_filename, log_filename = x
nc = max(1, num_cores / len(jobs))
# bowtie2 -p 8 -x <genome> -1 <.fq> -2 <.fq> --fr
# 2> test.log | samtools view -bS -o test.bam -
x1 = alignlib.make_bowtie2_command(ref.fasta_file_full,
j.temp_pair1,
fastq_file2=j.temp_pair2,
orientation=orientation,
num_threads=nc)
x2 = [
sq(samtools),
"view",
"-bS",
"-o",
sq(j.bam_filename),
"-",
]
x2 = " ".join(x2)
x = "%s 2> %s | %s" % (x1, sq(j.log_filename), x2)
#x = "%s >& %s" % (x, sq(log_filename))
commands.append(x)
metadata["commands"] = commands
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
x1 = [x.bam_filename for x in jobs]
x2 = [x.log_filename for x in jobs]
filelib.assert_exists_nz_many(x1 + x2)
return metadata
def run(
self, network, antecedents, out_attributes, user_options, num_cores,
out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, sai_node, orient_node, sample_node, reference_node = \
antecedents
fastq_files = mlib.find_merged_fastq_files(
sample_node.identifier, fastq_node.identifier)
sai_path = sai_node.identifier
assert filelib.dir_exists(sai_path)
orient = mlib.read_orientation(orient_node.identifier)
ref = alignlib.create_reference_genome(reference_node.identifier)
filelib.safe_mkdir(out_path)
metadata = {}
metadata["tool"] = "bwa %s" % alignlib.get_bwa_version()
# Technically, doesn't need the SampleGroupFile, since that's
# already reflected in the sai data. But better, because the
# sai data might not always be generated by BETSY.
# Find the merged fastq files.
# Find the sai files.
sai_filenames = filelib.list_files_in_path(
sai_path, endswith=".sai", case_insensitive=True)
assert sai_filenames, "No .sai files."
bwa = mlib.findbin("bwa")
# bwa samse -f <output.sam> <reference.fa> <input.sai> <input.fq>
# bwa sampe -f <output.sam> <reference.fa> <input_1.sai> <input_2.sai>
# <input_1.fq> <input_2.fq> >
# list of (pair1.fq, pair1.sai, pair2.fq, pair2.sai, output.sam)
# all full paths
jobs = []
for x in fastq_files:
sample, pair1_fq, pair2_fq = x
# The sai file should be in the format:
# <sai_path>/<sample>.sai Single end read
# <sai_path>/<sample>_1.sai Paired end read
# <sai_path>/<sample>_2.sai Paired end read
# Look for pair1_sai and pair2_sai.
pair1_sai = pair2_sai = None
for sai_filename in sai_filenames:
p, s, e = mlib.splitpath(sai_filename)
assert e == ".sai"
if s == sample:
assert not pair1_sai
pair1_sai = sai_filename
elif s == "%s_1" % (sample):
assert not pair1_sai
pair1_sai = sai_filename
elif s == "%s_2" % (sample):
assert not pair2_sai
pair2_sai = sai_filename
assert pair1_sai, "Missing .sai file: %s" % sample
if pair2_fq:
assert pair2_sai, "Missing .sai file 2: %s" % sample
if pair2_sai:
assert pair2_fq, "Missing .fq file 2: %s" % sample
sam_filename = os.path.join(out_path, "%s.sam" % sample)
log_filename = os.path.join(out_path, "%s.log" % sample)
x = sample, pair1_fq, pair1_sai, pair2_fq, pair2_sai, \
sam_filename, log_filename
jobs.append(x)
orientation = orient.orientation
#orientation = sample_node.data.attributes["orientation"]
assert orientation in ["single", "paired_fr", "paired_rf"]
# Make a list of bwa commands.
sq = mlib.sq
commands = []
for x in jobs:
sample, pair1_fq, pair1_sai, pair2_fq, pair2_sai, \
sam_filename, log_filename = x
if orientation == "single":
assert not pair2_fq
assert not pair2_sai
samse = "samse"
if orientation.startswith("paired"):
samse = "sampe"
x = [
sq(bwa),
samse,
"-f", sq(sam_filename),
sq(ref.fasta_file_full),
]
if orientation == "single":
x += [
sq(pair1_sai),
sq(pair1_fq),
]
else:
y = [
sq(pair1_sai),
sq(pair2_sai),
sq(pair1_fq),
sq(pair2_fq),
]
if orientation == "paired_rf":
y = [
sq(pair2_sai),
sq(pair1_sai),
sq(pair2_fq),
sq(pair1_fq),
]
x += y
x += [
">&", sq(log_filename),
]
x = " ".join(x)
commands.append(x)
metadata["commands"] = commands
metadata["num_cores"] = num_cores
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
x = [x[-2] for x in jobs]
filelib.assert_exists_nz_many(x)
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import genomelib
from genomicode import config
from Betsy import module_utils as mlib
fasta_node, bam_node, sample_node, orient_node = antecedents
fasta_data = mlib.find_merged_fastq_files(sample_node.identifier,
fasta_node.identifier,
find_fasta=True)
bam_filenames = mlib.find_bam_files(bam_node.identifier)
orient = mlib.read_orientation(orient_node.identifier)
filelib.safe_mkdir(out_path)
# TODO: Try to figure out version.
metadata = {}
metadata["tool"] = "RSeQC (unknown version)"
pyrseqc = mlib.findbin("pyrseqc")
gene_model = mlib.get_user_option(user_options,
"gene_model",
not_empty=True,
allowed_values=["hg19"])
if gene_model == "hg19":
gene_path = config.rseqc_hg19
else:
raise AssertionError, "Unhandled: %s" % gene_model
filelib.dir_exists(gene_path)
gene_model_bed = os.path.join(gene_path, "RefSeq.bed12")
housekeeping_model_bed = os.path.join(gene_path,
"HouseKeepingGenes.bed")
sample2fastadata = {}
for x in fasta_data:
sample, f1, f2 = x
sample2fastadata[sample] = x
is_paired = orient.orientation.startswith("paired")
# Guess the read length. Read the first fasta.
assert sample2fastadata
x = sample2fastadata.keys()[0]
filename = sample2fastadata[x][1]
lengths = {} # length -> count
for i, x in enumerate(genomelib.read_fasta_many(filename)):
if i >= 100:
break
title, sequence = x
l = len(sequence)
lengths[l] = lengths.get(l, 0) + 1
# Use the most common length.
c_length = c_count = None
for (l, c) in lengths.iteritems():
if c_count is None or c > c_count:
c_length, c_count = l, c
assert c_length
read_length = c_length
jobs = [] # sample, bam_filename, fasta_file1, fasta_file2, outdir
for bam_filename in bam_filenames:
# <path>/<sample>.bam
p, sample, e = mlib.splitpath(bam_filename)
assert sample in sample2fastadata
x, f1, f2 = sample2fastadata[sample]
outdir = os.path.join(out_path, sample)
x = sample, bam_filename, f1, f2, outdir
jobs.append(x)
# Some of the modules of RSeQC uses a lot of memory. Have
# seen a Python process take 33 Gb, and an R process take 200
# Gb. However, most of the modules use much less memory. So
# run one pyrseqc at a time, and run each one of those
# processes in parallel. Is probably slower than running
# multiple pyrseqc, but takes less memory.
commands = []
for x in jobs:
sample, bam_filename, fasta_filename1, fasta_filename2, outdir = x
# pyrseqc.py -j 20 --paired_end rqc11.bam rqc14.fa 76 \
# mod07.txt hg19.HouseKeepingGenes.bed rqc21 --dry_run
x = [
mlib.sq(pyrseqc),
"-j",
str(num_cores),
]
if is_paired:
x += ["--paired_end"]
x += [
mlib.sq(bam_filename),
mlib.sq(fasta_filename1),
str(read_length),
mlib.sq(gene_model_bed),
mlib.sq(housekeeping_model_bed),
mlib.sq(outdir),
]
x = " ".join(x)
commands.append(x)
metadata["commands"] = commands
metadata["num_cores"] = num_cores
# pyrseqc takes up to ~40 Gb per process.
# read_distribution.py takes 33 Gb.
# read_quality.py spins off an R process that takes ~200 Gb.
# Make sure we don't use up more memory than is available on
# the machine.
#nc = mlib.calc_max_procs_from_ram(60, upper_max=num_cores)
#metadata["num cores"] = nc
#x = parallel.pshell(commands, max_procs=nc)
# Because of memory, just run one at a time, but each one, use
# multiple cores.
for cmd in commands:
x = parallel.sshell(cmd)
assert x.find("Traceback") < 0, x
filelib.assert_exists_nz(out_path)
return metadata
def run(
self, network, antecedents, out_attributes, user_options, num_cores,
out_path):
import os
from genomicode import filelib
from genomicode import parallel
from Betsy import module_utils as mlib
# This this is I/O heavy, don't use so many cores. Also,
# takes 4-5 Gb RAM per process.
MAX_CORES = mlib.calc_max_procs_from_ram(5, upper_max=4)
fastq_node, sample_node, summary_node = antecedents
fastq_path = fastq_node.identifier
fastq_files = mlib.find_merged_fastq_files(
sample_node.identifier, fastq_path)
assert fastq_files, "I could not find any FASTQ files."
summary_filenames = filelib.list_files_in_path(
summary_node.identifier, endswith=".matches.txt")
assert summary_filenames, "No .matches.txt files."
filelib.safe_mkdir(out_path)
metadata = {}
num_mismatches = mlib.get_user_option(
user_options, "num_mismatches", type=int)
assert num_mismatches >= 0 and num_mismatches < 25
metadata["num_mismatches"] = num_mismatches
sample2summary = {} # sample -> summary_filename
for filename in summary_filenames:
# <sample>.matches.txt
p, f = os.path.split(filename)
assert f.endswith(".matches.txt")
sample = f.replace(".matches.txt", "")
assert sample not in sample2summary
sample2summary[sample] = filename
# list of (sample, fastq_file1, fastq_file2, summary_filename,
# out_file1, out_file2, subtracted_file1, subtracted_file2)
jobs = []
for x in fastq_files:
sample, pair1_fastq, pair2_fastq = x
assert sample in sample2summary, \
"Missing summary for sample: %s" % sample
p1, f1 = os.path.split(pair1_fastq)
if pair2_fastq:
p2, f2 = os.path.split(pair2_fastq)
assert p1 == p2
out1_fastq = os.path.join(out_path, f1)
sub1_fastq = os.path.join(out_path, "%s.subtracted" % f1)
out2_fastq = None
sub2_fastq = None
if pair2_fastq:
out2_fastq = os.path.join(out_path, f2)
sub2_fastq = os.path.join(out_path, "%s.subtracted" % f2)
x = sample, pair1_fastq, pair2_fastq, sample2summary[sample], \
out1_fastq, out2_fastq, sub1_fastq, sub2_fastq
jobs.append(x)
jobs2 = [] # list of (function, args, keywds)
for x in jobs:
sample, pair1_fastq, pair2_fastq, summary_file, \
out1_fastq, out2_fastq, sub1_fastq, sub2_fastq = x
x = summary_file, pair1_fastq, out1_fastq, sub1_fastq, \
num_mismatches
x = subtract_mouse_reads, x, {}
jobs2.append(x)
if pair2_fastq:
x = summary_file, pair2_fastq, out2_fastq, sub2_fastq, \
num_mismatches
x = subtract_mouse_reads, x, {}
jobs2.append(x)
nc = min(MAX_CORES, num_cores)
results = parallel.pyfun(jobs2, num_procs=nc, DELAY=0.5)
assert len(results) == len(jobs2)
metadata["num_cores"] = nc
# Make sure the fastq files were generated.
x1 = [x[4] for x in jobs]
x2 = [x[5] for x in jobs]
x = x1 + x2
x = [x for x in x if x]
# BUG: If all reads were removed, then this will fail incorrectly.
filelib.assert_exists_nz_many(x)
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import parallel
from genomicode import filelib
from genomicode import alignlib
from Betsy import module_utils as mlib
fastq_node, group_node, reference_node = antecedents
fastq_files = mlib.find_merged_fastq_files(group_node.identifier,
fastq_node.identifier)
assert fastq_files, "No FASTQ files found."
ref = alignlib.create_reference_genome(reference_node.identifier)
filelib.safe_mkdir(out_path)
metadata = {}
metadata["tool"] = "bwa %s" % alignlib.get_bwa_version()
# Make sure no duplicate samples.
x1 = [x[0] for x in fastq_files]
x2 = {}.fromkeys(x1).keys()
assert len(x1) == len(x2), "dup sample"
# Make a list of all FASTQ files to align.
fastq_filenames = []
for x in fastq_files:
sample, pair1, pair2 = x
assert pair1
fastq_filenames.append(pair1)
if pair2:
fastq_filenames.append(pair2)
# Make a list of all the jobs to do.
jobs = [] # list of (fastq_filename, sai_filename)
for in_filename in fastq_filenames:
in_path, in_file = os.path.split(in_filename)
x = in_file
if x.lower().endswith(".fq"):
x = x[:-3]
elif x.lower().endswith(".fastq"):
x = x[:-6]
sai_filename = os.path.join(out_path, "%s.sai" % x)
log_filename = os.path.join(out_path, "%s.log" % x)
x = in_filename, sai_filename, log_filename
jobs.append(x)
# Calculate the number of threads per job.
nc = max(1, num_cores / len(jobs))
# Make the bwa commands.
commands = []
for x in jobs:
fastq_filename, sai_filename, log_filename = x
x = alignlib.make_bwa_aln_command(ref.fasta_file_full,
fastq_filename,
sai_filename,
log_filename,
num_threads=nc)
commands.append(x)
metadata["commands"] = commands
metadata["num cores"] = num_cores
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
for x in jobs:
in_filename, sai_filename, log_filename = x
assert filelib.exists_nz(sai_filename), \
"Missing: %s" % sai_filename
return metadata
def run(self, network, antecedents, out_attributes, user_options,
num_cores, out_path):
import os
from genomicode import filelib
from genomicode import parallel
from Betsy import module_utils as mlib
fastq_node, sample_node = antecedents
fastq_files = mlib.find_merged_fastq_files(sample_node.identifier,
fastq_node.identifier)
assert fastq_files, "I could not find any FASTQ files."
filelib.safe_mkdir(out_path)
metadata = {}
adapters_filename = mlib.get_user_option(user_options,
"adapters_fasta",
not_empty=True,
check_file=True)
if " " in adapters_filename:
os.symlink(adapters_filename, "adapters.txt")
adapters_filename = "adapters.txt"
jobs = []
for x in fastq_files:
sample, pair1, pair2 = x
p1, f1 = os.path.split(pair1)
trimmed1 = os.path.join(out_path, f1)
trimmed2 = None
if pair2:
p2, f2 = os.path.split(pair2)
trimmed2 = os.path.join(out_path, f2)
# BUG: Will be overwritten. Need to give unpaired files
# unique names.
unpaired1 = os.path.join(out_path, "unpaired_1.fasta")
unpaired2 = os.path.join(out_path, "unpaired_2.fasta")
log_filename = os.path.join(out_path, "%s.log" % sample)
x = sample, pair1, pair2, trimmed1, trimmed2, \
unpaired1, unpaired2, log_filename
jobs.append(x)
sq = parallel.quote
commands = []
for x in jobs:
sample, pair1, pair2, trimmed1, trimmed2, unpaired1, unpaired2, \
log_filename = x
nc = max(1, num_cores / len(jobs))
x = _make_trimmomatic_cmd(pair1,
pair2,
trimmed1,
trimmed2,
unpaired1,
unpaired2,
adapters_filename,
num_threads=nc)
x = "%s >& %s" % (x, sq(log_filename))
commands.append(x)
metadata["commands"] = commands
parallel.pshell(commands, max_procs=num_cores)
# Make sure the analysis completed successfully.
for x in jobs:
sample, pair1, pair2, trimmed1, trimmed2, unpaired1, unpaired2, \
log_filename = x
# Make sure outfile created.
assert filelib.exists_nz(trimmed1), \
"Missing: %s" % trimmed1
if trimmed2:
assert filelib.exists_nz(trimmed2), \
"Missing: %s" % trimmed2
x = open(log_filename).read()
assert not x.startswith("Usage:"), "usage problem"
return metadata